The separation of chondroitin sulfate disaccharides and hyaluronan oligosaccharides by capillary zone electrophoresis

We have developed techniques for the separation of unsulfated (2-acetamido-2-deoxy-3-O-(4-deoxy-alpha-L-threo- hex-4-enopyranosyluronicacid)-D-galactose and -D-glucose), monosulfated (2-acetamido-2-deoxy-3- O-(4-deoxy-2-O-sulfo-alpha-L-threo-hex-4-enopyranosyluronic acid)-D-galactose and 2-acetamido-2-deoxy-3-O-(4-deoxy-alpha-L-threo-hex- 4-enopyranosyluronic acid)-4-sulfo-D-galactose and -6-sulfo-D-galactose),disulfated (2-acetamido-2-deoxy-3-O-(4-deoxy-2-O-sulfo-alpha-L-threo-hex-4- enopyranosyluronic acid)-4-sulfo-D-galactose and -6-sulfo-D-galactose and 2-acet-amido-2-deoxy-3-O-(4-deoxy-alpha-L-threo-hex-4-enopy- ranosyluronic acid)-4,6-di-O-sulfo-D-galactose), and trisulfated (2-acetamido-2-deoxy-3-O-(4-deoxy-2-O- sulfo-alpha-L-threo-hex-4-enopyranosyluronic acid)-4,6-di-O-sulfo-D-galactose) isomers of chondroitin using capillary zone electrophoresis. In addition, it is possible to separate oligomers of hyaluronan by similar protocols. These techniques represent a rapid, sensitive, and reproducible technique for the assay of these molecules from digests of connective tissues.     

Comparison of pregnancy rates from transfer of fresh versus cooled, transported equine embryos

Donor mares of mixed, light-horse breeds, maintained at Colorado State University, provided 104 embryos for immediate transfer (fresh embryos). One hundred and thirty-six additional embryos were collected on various breeding farms in the United States and were shipped to Colorado State University via commercial airlines (cooled embryos). Embryos were harvested 7 d after ovulation, graded, and either transferred into a mare immediately (<1 h) or placed in Ham's F-10 medium plus 10% fetal calf serum in an atmosphere of 5% CO(2), 5% O(2), 90% N(2) and packaged in a passive cooling unit (Equitainer) for shipment to our laboratory. All embryos were measured and graded just prior to surgical transfer via flank incision into synchronized mares. Recipients had ovulated 1 or 2 d before (+1, +2), on the same day as (0), or 1, 2 or 3 d after (-1, -2, -3) the donor mare. Pregnancy of recipients was determined by ultrasonography on 12, 35, and 50 d after ovulation of the donor. Pregnancy rates at 12, 35, and 50 d were similar for fresh (74, 64, 61%) and cooled embryos (80, 67, 66%), respectively. Overall, embryo size affected (P<0.05) pregnancy rates at 12, 35 and 50 d. Embryos of Grade 1 (excellent) or 2 resulted in more pregnancies than those of Grade 3 or 4 (poor) embryos. Embryonic losses between 12 and 35 d or between 35 and 50 d were not altered (P>0.05) by treatment (fresh or cooled) nor by age of the donor mare (P>0.05), but embryonic losses between 12 and 35 d were greater (P<0.06) for embryos stored for >12 h (25%) versus those stored for <12 h (10%). The duration needed for shipment (<12 h or >12 h) of cooled embryos did not alter pregnancy rates at 12 d (P>0.05). Age of donor mare had no effect (P>0.05) upon pregnancy rates of cooled or fresh embryos transferred nor on embryo quality. In summary, equine embryos can be cooled to 5 degrees C and maintained in storage for up to 24 h without decreased fertility, compared with those of embryos transferred in <1 hour.     

Improved development of rabbit one-cell embryos to the hatching blastocyst stage by culture in a defined, protein-free culture medium

In Exp. 1, Medium 199 and Medium RD (RPMI-1640 and Dulbecco's MEM, 1:1 v/v) were compared in a 2 x 2 factorial design by supplementing each with 15 mg bovine serum albumin (BSA)/ml of 1 mg polyvinyl alcohol (PVA)/ml. All media contained 5 micrograms insulin/ml, 5 micrograms transferrin/ml, 5 ng selenium/ml (ITS), and 10 ng epidermal growth factor (EGF)/ml. One-cell embryos were cultured at 39 degrees C with 5% CO2 in air for 65 h and then stained with Hoechst 33342 to determine blastomere number. Embryos in Medium 199 developed poorly (P less than 0.001) when PVA was used instead of BSA (30 vs 76 cells/embryo), but developed rapidly in Medium RD with PVA or BSA (118 and 121 cells). Similar results were obtained in Exp. 2 in BSA- and PVA-free medium. In Exp. 3, the development of 1-cell embryos after 65 h in unsupplemented (protein-free) Medium RD (68% blastocysts, 117 cells) did not differ (P greater than 0.37) from that obtained using Medium RD with insulin, ITS or EGF alone. Culture in protein-free Medium RD for 96 h resulted in 82% of the 1-cell embryos forming blastocysts and 40% hatching through the zona pellucida. In a preliminary test of viability, 1-cell embryos cultured in this medium for 48 or 65 h and transferred to synchronous recipients resulted in 5/18 (28%) and 3/24 (12%) Day-15 viable fetuses. Cell counts of approximately 120 per blastocyst after culturing 1-cell embryos for 65 h in Medium RD indicated that cell division was more rapid than that obtained with all other media tested previously in this laboratory. This is the first report of rabbit embryo development from the 1-cell to the hatching blastocyst stage in a defined protein-free culture medium.     

Bovine 1-2-cell embryo development using a simple medium in three oviduct epithelial cell coculture systems

These studies were designed to develop a coculture system using a simple medium to promote development of 1-cell bovine embryos through the 8-16-cell stage to morula and blastocyst stages. Monolayers for coculture were prepared from bovine oviduct epithelial cells (BOEC). In vivo-fertilized 1-2-cell embryos and ova (384) were surgically collected from superovulated cows. In Experiment 1, embryos cocultured in a simple glucose-free and serum-free medium (CZB) developed with superior scores of embryo quality than embryos cocultured in Ham's F-10 with serum, and a greater percentage developed past 8-16 cells than embryos cocultured in CMRL-1066 with serum (p less than 0.05). In Experiment 2, embryos cocultured with fresh BOEC monolayers averaged more (p less than 0.05) cells than did embryos in coculture with frozen-thawed BOEC monolayers or in BOEC-conditioned medium. Without glucose in the simple medium for the first 48 h of culture, more embryos blastulated (p less than 0.01) by Day 5.5 of culture (Day 6.5 of donor's estrous cycle) than embryos in the same medium with glucose present throughout. In Experiment 3, more embryos tended to hatch in BOEC coculture (p less than 0.10) than in conditioned medium. These results show that a chemically simple medium with fresh BOEC monolayers can provide a significant benefit for coculture of early bovine embryos.     

Seasonal Occurrence and Distribution of Microbial Indicators and Pathogens in the Rhode River of Chesapeake Bay

The seasonal incidence and occurrence of indicator organisms and pathogens were studied at four sites in the Rhode River, a subestuary of Chesapeake Bay. The highest frequency of occurrence of total and fecal coliforms and fecal streptococci was in Muddy Creek, a marsh area receiving pasture land runoff. Second highest frequency of occurrence of these bacteria was in Cadle Creek, a populated area. Lowest measurements of these parameters were obtained at stations in the central portion of the Rhode River. No Salmonella spp. were detected by the methods employed in this study. However, it is concluded that if these organisms are present, the concentrations are ≤1 organism per liter. The presence of Clostridium botulinum was detected in 12% of the samples tested.     

Phosphoinositides in mitogenesis: neomycin inhibits thrombin-stimulated phosphoinositide turnover and initiation of cell proliferation

Thrombin stimulates 32Pi incorporation into phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bis-phosphate (PIP2), and phosphatidylinositol (PI), and initiates DNA synthesis in hamster (NIL) fibroblasts at a half-maximal concentration of 125 ng/ml. Neomycin, which binds PIP2 and PIP, inhibits both thrombin-stimulated initiation of cell proliferation and 32P pI incorporation into at concentrations above 2 mM without affecting thrombin binding, thymidine uptake, or cellular protein synthesis. At lower concentrations, neomycin inhibits thrombin-stimulated release of inositol 1,4,5-trisphosphate (IP3), by selectively binding PIP2, but does not inhibit 32P incorporation into PI or initiation of DNA synthesis. Phosphoinositide recycling and diacylglycerol release therefore appear necessary for initiation of cell proliferation by thrombin. IP3-stimulated Ca++ mobilization may not be required for thrombin mitogenesis, however, since neomycin can block IP3 release without inhibiting initiation.     

Adenylate cyclase activity and motor behavior following cerebral ischemia in the unanesthetized gerbil

Five minutes of bilateral carotid occlusion in unanesthetized gerbils produced substantial changes in spontaneous locomotor activity. Behavior was decreased after 1 hr of reperfusion and was increased at 24 hrs post-ischemia. Adenylate cyclase activity was measured in homogenates of frontal cortex and hippocampus at 90 min and 24 hrs following 5 min of cerebral ischemia. Enzyme activity was determined in the absence and presence of the activators guanosine-5'-triphosphate (GTP), guanylyl-5'-imidodiphosphate (GppNHp), isoproterenol (Iso) plus GTP, and forskolin (Fors) plus GTP. Homogenates responded with expected increases over basal adenylate cyclase activity with addition of all activators. An additional small increase in isoproterenol-stimulated activity was observed in frontal cortex homogenates at 90 min post-ischemia. No other significant changes in adenylate cyclase activity were observed after either 90 min or 24 hrs of reperfusion. The substantial increases in locomotor activity evident at 24 hrs after transient ischemia are not associated with measurable changes in adenylate cyclase activity in homogenates of frontal cortex or hippocampus.     

Identification of an arginine vasopressin-like diuretic hormone from Locusta migratoria

We have identified two neuropeptides (F1 and F2) from suboesophageal and thoracic ganglia of Locusta migratoria, which we isolated earlier based on their immunological similarity to arginine vasopressin. The more abundant and hydrophilic factor, F1, has sequence Cys-Leu-Ile-Thr-Asn-Cys-Pro-Arg-Gly-NH2, but its biological role is unknown. The less abundant factor, F2, is an antiparallel dimer of F1, and functions as a diuretic hormone of this species. It appears to act through the intermediacy of cyclic AMP. The properties of the native neuropeptides were identical with those of samples synthesized from appropriately protected L-amino acids.     

The susceptibility of muscle phosphorylases a and b to digestion by a neutral proteinase from rat intestinal muscle. Comparison with the effects produced by pancreatic trypsin and chymotrypsin

1. Phosphorylase b was inactivated three times more rapidly than phosphorylase a by a neutral, trypsin-like proteinase from rat intestinal muscle. Digestion of phosphorylase a produced a modified form which was deactivated by AMP. Removal of the pyridoxal phosphate cofactor increased the rate of inactivation of the b form by about 3-fold but the subceptibility of apophosphorylase a was no different from the holo form. 2. The extent of proteolysis of both holoenzyme forms, as guaged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, was limited and similar digestion patterns were obtained in both cases. 3. With ³²P-labelled phosphorylase a as substrate, the initial event in the inactivation was the release of a trichloroacetic acid-soluble peptide from the N-terminus of the enzyme, leaving the original 100000 subunit form essentially unchanged. Subsequent proteolysis was restricted, producing derivatives of mol.wt. 85000, 70000 and 65000, none of which contained any radioactive label. 4. By treatment of inactivated phosphorylase b with carboxypeptidase B, it was shown that the intestinal muscle proteinase had cleaved approximately 3 -Lys-X and 3 -Arg-X bonds in the polypeptide. 5. The protective effects of various allosteric modulators of phosphorylase on the inactivation of the a and b forms were generally in agreement with the known roles of the modifiers. Glucose increased the susceptibility of phosphorylase a. 6. Inactivation of phosphorylase b by trypsin and chymotrypsin also resulted in limited proteolysis but, in both cases, the digestion patterns obtained on sodium dodecyl sulphate/polyacrylamide gels were different from each other and from the pattern obtained with the intestinal muscle proteinase. 7. Inactivation of phosphorylase b by the muscle proteinase is about 100 times more rapid than the effects produced by trypsin or chymotrypsin when the activities are compared on an equimolar basis. 8. Consideration is given to regulation of the rate of enzyme degradation intracellularly by modulation of the conformation and susceptibility of the enzyme via factors such as covalent modification, allosteric ligands and state of aggregation.